Breast cancer is the second leading cause of cancer-related deaths among women, with deaths occurring due to the metastatic spread of tumor cells to distant organs. Spread of cancer cells occurs via blood vessels or, particularly important for breast cancer, via the lymphatic system. Although tumor dissemination via the blood has been extensively studied, the mechanisms that lead to lymphatic dissemination are poorly understood. This proposal focuses on understanding a new mechanism of lymphangiogenesis and breast tumor metastatic spread, via control of VEGF-C levels by the Six1/Eya2 transcriptional complex. The hypothesis to be tested is that Six1 overexpression in breast cancers leads to upregulation of VEGF-C, resulting in increased lymphangiogenesis and enhancing the early stages of metastasis including lymphatic dissemination of tumor cells. The ability of Six1 to activate VEGF-C and stimulate lymphatic metastasis is dependent on its interaction with the Eya2 phosphatase co-factor, and on the enzymatic activity of Eya2. If this hypothesis is correct, it will provide important insights into the mechanism of breast cancer metastasis and provide a rationale for a new strategy to therapeutically target lymphatic metastasis by interfering with Six1/Eya2 function through the druggable approaches of inhibiting Six1/Eya interaction and/or Eya2 phosphatase activity. To address this hypothesis, we will: 1) Test the hypothesis that VEGF-C is a direct transcriptional target of Six1/Eya2, and that Six1 and Eya2 correlate with VEGF-C and lymphangiogenesis in human breast cancer; 2) Test the hypothesis that VEGF-C is a critical mediator of Six1-induced lymphangiogenesis and metastasis using in vivo mouse metastasis models; and 3) Test the hypothesis that Eya2 is required for Six1-induced lymphangiogenesis and metastasis in vivo, with a specific emphasis on the interaction between Six1 and Eya2, as well as the role of the Eya2 phosphatase activity. Targeting Six1/Eya2 has the potential to inhibit breast cancer both at early (studied in this proposal) and later stages of the disease. Due to the paucity of expression of the Six1/Eya2 developmental regulators in most normal adult tissues, and their re-expression in cancers, therapeutic agents targeting this complex should inhibit lymphangiogenesis and metastasis with limited side effects. Thus the Six1/Eya2 complex is an ideal breast cancer therapeutic target, and work within this proposal will lay the foundation for eventual targeting of the complex.